Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration

Abstract Background The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP) activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. Methods The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. Results The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only ~30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol), which lacks telopeptide regions. Conclusion Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis

Cell–cell and cell–matrix dynamics in intraperitoneal cancer metastasis

The peritoneal metastatic route of cancer dissemination is shared by cancers of the ovary and gastrointestinal tract. Once initiated, peritoneal metastasis typically proceeds rapidly in a feed-forward manner. Several factors contribute to this efficient progression. In peritoneal metastasis, cancer cells exfoliate into the peritoneal fluid and spread locally, transported by peritoneal fluid. Inflammatory cytokines released by tumor and immune cells compromise the protective, anti-adhesive mesothelial cell layer that lines the peritoneal cavity, exposing the underlying extracellular matrix to which cancer cells readily attach. The peritoneum is further rendered receptive to metastatic implantation and growth by myofibroblastic cell behaviors also stimulated by inflammatory cytokines. Individual cancer cells suspended in peritoneal fluid can aggregate to form multicellular spheroids. This cellular arrangement imparts resistance to anoikis, apoptosis, and chemotherapeutics. Emerging evidence indicates that compact spheroid formation is preferentially accomplished by cancer cells with high invasive capacity and contractile behaviors. This review focuses on the pathological alterations to the peritoneum and the properties of cancer cells that in combination drive peritoneal metastasis

Dynamic distribution of nuclear coactivator 4 during mitosis: association with mitotic apparatus and midbodies.

The cytoplasmic localization of Nuclear Receptor Coactivator 4 (NcoA4), also referred to as androgen receptor associated protein 70 (ARA70), indicates it may possess activities in addition to its role within the nucleus as a transcriptional enhancer. Towards identifying novel functions of NcoA4, we performed an in silico analysis of its amino acid sequence to identify potential functional domains and related proteins, and examined its subcellular distribution throughout the cell cycle. NcoA4 has no known or predicted functional or structural domains with the exception of an LxxLL and FxxLF nuclear receptor interaction motif and an N-terminal putative coiled-coil domain. Phylogenetic analysis indicated that NcoA4 has no paralogs and that a region referred to as ARA70-I family domain, located within the N-terminus and overlapping with the coiled-coil domain, is evolutionarily conserved in metazoans ranging from cnidarians to mammals. An adjacent conserved region, designated ARA70-II family domain, with no significant sequence similarity to the ARA70-I domain, is restricted to vertebrates. We demonstrate NcoA4 co-localizes with microtubules and microtubule organizing centers during prophase. Strong NcoA4 accumulation at the centrosomes was detected during interphase and telophase, with decreased levels at metaphase and anaphase. NcoA4 co-localized with tubulin and acetylated tubulin to the mitotic spindles during metaphase and anaphase, and to midbodies during telophase. Consistent with these observations, we demonstrated an interaction between NcoA4 and α-tubulin. Co-localization was not observed with microfilaments. These findings indicate a dynamic distribution of NcoA4 with components of the mitotic apparatus that is consistent with a potential non-transcriptional regulatory function(s) during cell division, which may be evolutionarily conserved

Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration

Abstract Background The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP) activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. Methods The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. Results The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only ~30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol), which lacks telopeptide regions. Conclusion Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis.</p

Ventricular Zone Expressed PH Domain Containing 1 (VEPH1): an adaptor protein capable of modulating multiple signaling transduction pathways during normal and pathological development

Abstract Ventricular Zone Expressed PH Domain-Containing 1 (VEPH1) is an 833-amino acid protein encoded by an evolutionarily conserved single-copy gene that emerged with pseudocoelomates. This gene has no paralog in any species identified to date and few studies have investigated the function of its encoded protein. Loss of expression of its ortholog, melted, in Drosophila results in a severe neural phenotype and impacts TOR, FoxO, and Hippo signaling. Studies in mammals indicate a role for VEPH1 in modulating TGFβ signaling and AKT activation, while numerous studies indicate VEPH1 expression is altered in several pathological conditions, including cancer. Although often referred to as an uncharacterized protein, available evidence supports VEPH1 as an adaptor protein capable of modulating multiple signal transduction networks. Further studies are required to define these adaptor functions and the role of VEPH1 in development and disease progression

NcoA4 localization and preabsorption with corresponding immunizing peptide and localization of ECFP-tagged NcoA4 at the mitotic spindle.

<p>COS cells were stained for NcoA4 (red) using a goat polyclonal antibody (Q19) and examined by confocal microscopy. Cells were also subjected to DAPI staining for visualization of chromatin (blue). Shown is a representative image of a cell undergoing metaphase (<b>A</b> to <b>C</b>). Immunofluorescence with the antibody preabsorbed with the corresponding immunizing peptide (<b>D</b> to <b>I</b>). COS cells transiently transfected with pECFP-NcoA4 cDNA and stained for CFP-NcoA4 fusion protein using CFP antibody (<b>J</b> to <b>L</b>). All images shown are of single optical slices. [Bars are 2.7 µm to 3.9 µm as indicated].</p

Co-localization of NcoA4 and α-tubulin in COS cells treated with nocodazole.

<p>COS cells untreated (<b>A</b> to <b>D</b>) or treated with 100 ng/ml nocodazole for 12 hours (<b>E</b> to <b>L</b>) were stained for NcoA4 (red) and α-tubulin (green) and examined by confocal microscopy. Cells were also subjected to DAPI staining for visualization of cell nuclei (blue). Yellow staining indicates NcoA4-α-tubulin co-localization. Images shown are of single optical slices. Arrows indicate mitotic figures. [Bars are 1.7 µm-9 µm as indicated].</p

Co-localization of NcoA4 with tubulin and actin.

<p>T47D (<b>A</b> to <b>C</b> and <b>G</b> to <b>I</b>) or COS (<b>D</b> to <b>F</b> and <b>J</b> to <b>L</b>) cells were stained for NcoA4 and either α-tubulin (<b>A</b> to <b>C</b>), β-tubulin (<b>D</b> to <b>F</b>), acetylated tubulin (<b>G</b> to <b>I</b>) or actin (<b>J</b> to <b>L</b>). Cells were subjected to DAPI staining for visualization of chromatin (blue). Shown are representative images of non-mitotic cells (<b>A</b> to <b>C</b>, yellow arrow) and cells undergoing cell division (prophase: <b>A</b> to <b>C</b>, metaphase: <b>D</b> to <b>I</b>, and anaphase: <b>J</b> to <b>L</b>, white arrows). An aster formation is shown in <b>D</b> to <b>F</b> (red arrow). Yellow staining indicates overlapping NcoA4 and tubulin or actin (inset; orange arrow) localization. All images shown are of single optical slices obtained by confocal (<b>A</b> to <b>C</b> and <b>G</b> to <b>I</b>) or deconvolution (<b>D</b> to <b>F</b> and <b>J</b> to <b>L</b>) microscopy. [Bars are 1.7 µm-20 µm as indicated].</p

NcoA4 association with the centrosome during mitotic progression.

<p>Representative images of COS cells at different stages of mitosis stained for NcoA4 (red; <b>A,</b> and <b>D</b>), and γ−tubulin (green; <b>B</b>), or Plk1 (green; <b>E</b>). Merged images are shown in <b>C</b> and <b>F</b>. All images shown are of single optical slices obtained by confocal (<b>A</b> to <b>C</b>) or deconvolution (<b>D</b> to <b>F</b>) microscopy. Cells were also subjected to DAPI staining for visualization of chromatin (blue). NcoA4 was localized to aster formations (yellow arrows, <b>A</b> and <b>C</b>) and centrosomes (white arrows, <b>A</b> and <b>C</b>). Diminished NcoA4 staining at the centrosome during metaphase (red arrows, <b>A</b> and <b>D</b>) using γ-tubulin (<b>C</b>) or Plk1 (<b>F</b>) as a centrosomal marker. [Bars: 20 µm].</p